One of the defense mechanisms against infection by both animals and plants is the production of peptides that have antimicrobial and antiviral activity. Various classes of these peptides have been isolated from tissues of plants and animals. These classes include the tachyplesins (Nakamura et al., 1988), the xe2x80x9cprotegrinsxe2x80x9d (Kokryakov et al., 1993) and the defensins, which include beta-defensins and classical defensins. The defensins are characterized by six invariant cysteines and three intramolecular cystine disulfide bonds (Lehrer et al., 1991; Lehrer et al., 1993). Classical defensins are short (29-35 amino acid residues) cationic peptides containing three pair of disulfide-linked cysteines. Although beta-defensins are nearly identical in their three-dimensional structure to classical defensins, beta-defensins are slightly larger (38-42 amino acid residues) and differ in the spacing of the conserved cysteine residues and connectivity of the cysteine linkages relative to classical defensins (Zimmerman et al., 1995).
Heterophils are the major granulated leukocyte of birds and are similar to the mammalian neutrophil. Avian heterophils lack myeloperoxidase and alkaline phosphatase activities but their ultrastructure, cytochemistry, and functions are otherwise similar to mammalian neutrophils (Pennial and Spitznagel, 1975; Montali, 1988). Both heterophils and neutrophils possess cationic antimicrobial peptides, which are important mediators of innate disease resistance in tissues exposed to microbial pathogens. Cationic antimicrobial peptides likely exert their antimicrobial activity by interacting with cell membranes or viral envelopes initially via electrostatic forces then by membrane insertion in which they form voltage gated ion channels resulting in increased permeability (Boman, 1991; Kagan et al., 1990).
Numerous mammalian neutrophil classical defensins have been isolated and sequenced (Selsted and Harwig, 1987; Belcourt et al., 1992; Wilde et al., 1989; U.S. Pat. No. 5,202,420). Three chicken heterophil beta-defensin mature peptide amino acid sequences (Gal 1/CHP 1, Gal 1xcex1/CHP 2, and Gal 2) (Harwig et al., 1994; Evans et al., 1994; U.S. Pat. No. 5,202,420) and three partial turkey heterophil beta-defensins (THP 1, THP 2, and THP 3) have been reported (Evans et al., 1994). These avian beta-defensins are bacteriocidal in vitro for both avian and human bacterial pathogens (Evans et al., 1995). Thirteen mature bovine neutrophil beta-defensins amino acid sequences have also been reported (Selsted et al., 1993; U.S. Pat. No. 5,459,235).
Defensins are synthesized as 93-95 residue prepro defensins with a hydrophobic 19 amino acid signal sequence which is necessary for insertion into the endoplasmic reticulum prior to transport to granules. It has been proposed that neutrophil storage granule peptides (classical defensins) have a negatively charged propiece (of about 40-45 amino acids) to neutralize or balance the positive charge of the mature peptide (Michaelson et al., 1992). This propiece also is necessary for proper peptide folding and in targeting of the mature peptide into storage granules (Liu and Ganz, 1995). Thus far, all characterized classical defensins from mammalian granulocytes and from intestinal Paneth cells have a negatively charged propiece (Jones and Bevins, 1993; Yount et al., 1995).
In contrast, storage granule-free epithelial cell beta-defensin propeptides of the respiratory and oral cavity, i.e., bovine tracheal antimicrobial peptide (TAP) and lingual antimicrobial peptide (LAP), have an abridged propiece that has no neutralizing negative charge (Diamond et al., 1991; Schonwetter et al., 1995). These antimicrobial peptides are synthesized de novo upon stimulation and are not stored in granules (Russell et al., 1996; Schonwetter et al., 1995). The propieces for beta-defensins found in bovine neutrophils have not been characterized (Selsted et al., 1993).
Thus, what is needed is the identification and isolation of a variety of genes encoding avian peptides having specific antimicrobial activities.
The invention provides an isolated and purified nucleic acid molecule comprising a nucleic acid sequence which encodes an avian beta-defensin, a biologically active fragment or a biologically active variant thereof. As described hereinbelow, the complete cDNA for two chicken (Gal 1/CHP 1 and Gal 2) and two turkey (THP 1 and THP 2) beta-defensins were obtained. Surprisingly, the four deduced beta-defensin pro regions lack the long, negatively charged propiece reported for classical defensin pro regions, a region which is thought to stabilize and inactivate the positively charged mature peptide and target the propeptide to the storage granule.
Preferred isolated nucleic acid molecules of the invention include those having a nucleic acid sequence comprising a full-length avian beta-defensin gene, i.e., encoding the prepro form of the peptide (i.e., one having the signal sequence and the propiece), e.g., a nucleic acid sequence comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or a variant thereof, or comprising a nucleic acid sequence encoding the mature form of an avian beta-defensin, such as a nucleic acid sequence comprising SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or a variant thereof. Also preferably, the nucleic acid molecules of the invention encode a peptide having an amino acid corresponding to the prepro form of an avian beta-defensin, e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or a variant thereof, or an amino acid sequence corresponding to the mature form of an avian beta-defensin, such as SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or a variant thereof. It is preferred that the nucleic acid molecules of the invention encode a peptide having antimicrobial (e.g., antibacterial, antiviral, antiprotozoal or antifungal) activity, more preferably peptides having a broad spectrum (for example, they are active against a variety of bacteria of different genera) of antimicrobial activity.
It is also envisioned that anti-sense nucleic acid molecules, e.g., a molecule which is the complement of an avian beta-defensin, a biologically active fragment, variant thereof, are within the scope of the invention. The nucleic acid molecules of the invention, fragments or variants thereof, either DNA or RNA, are useful to prepare probes, primers or expression cassettes which, in turn, are useful to detect, amplify and express other avian beta-defensin genes and related genes.
Therefore, the invention also provides an expression cassette comprising: a DNA sequence which is operably linked to a promoter functional in a host cell, which DNA sequence encodes an avian beta-defensin, a biologically active fragment or a biologically active variant thereof. The host cell may be prokaryotic or eukaryotic in origin. These cassettes may be employed to prepare recombinant peptides. For example, an expression cassette of the invention may be introduced and expressed in a host cell, e.g., an insect cell using a baculovirus vector, so as to yield recombinant avian beta-defensin peptide, a biologically active fragment, or variant thereof. Preferably, the recombinant peptide is recovered from the host cell. It is preferred that a peptide of the invention is active against at least one pathogen including, but not limited to, Staphylococcus aureus, Escherichia coli, Pasteurella multocida, Bordetella avium, Mycoplasma gallispeticum, Candida albicans, Listeria monocytogenes, Salmonella typhimurium, Salmonella enteriditis, or Campylobacter jejuni. 
Hence, the invention further provides an isolated and purified avian beta-defensin peptide, a biologically active variant or fragment thereof. Preferred peptides include the prepro form of an avian beta-defensin. More preferably, the peptide is a compound of formula (I):
GCPSX2X3X3X2X3GSCFGFX2SCCX2WPWNX3, SEQ ID NO:49 wherein X3 is I, V, M, A, norleucine or L, wherein X2 is H, N, Q, K or R, and wherein X1 is I, L or V;
a compound of formula (II):
MRX1VYX1X1X1PFX1X1X1X1AQGAAGSSQAX1GX2X2SX1CFX2X2SGFCAFX3KCPSX3TX3X3SGKCSX2FYX3CCX2X2X3WG, SEQ ID NO:50 wherein X3 is I, V, M, A, norleucine or L, wherein X2 is H, N, Q, K or R, and wherein X1 is I, L, or
a compound of formula (III):
MRX1X1YX1X1FPFX1X1X1X1AQGAAGSSX1AX1GX2X2EX2CX3X2X2X2GFCX3FX3X2CPTX3SX3X3SGTCSX2FX2X3CCX2T X3X3G, SEQ ID NO:51 wherein X3 is I, V, M, A, norleucine or L, wherein X2 is H, N, Q, K or R, and wherein X1 is I, L or V; or
a compound of formula (IV):
MRX1X1YX1X1FSX1X1FX1AX1Q X1PGX1SSPNRDMX3FCX2X2GTCX2FGX2CPSX2X3X3KX3GSCFGFX2SCCX2WPWDX3, S SEQ ID NO:52 wherein X3 is I, V, M, A, norleucine or L, wherein X2 is H, N, Q, K or R, and wherein X1 is I, L or V.
The recombinant peptide may be recovered from cell lysates in a soluble fraction, in the insoluble fraction, or from supernatants, i.e., the recombinant peptide is secreted into the extracellular environment of the host cell. Another embodiment of the invention is a fusion polypeptide comprising at least a portion of the avian beta-defensin peptide and at least a portion of a second polypeptide. These peptides are useful as antibiotic, disinfectants, antimicrobials in biological products, food preservatives or to treat food products, such as crops, shell fish and poultry, to eliminate potential pathogens. The peptides may also promote wound healing and tissue repair. Preferably, the isolated peptides of the invention have substantially the same antimicrobial activity as a peptide having SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or a variant thereof. These isolated peptides, or nucleic acid molecules encoding them, may also be useful to study microbial resistance to heterophil microbicidal activity, and the relationship between disease resistance and a particular avian beta-defensin peptide. Further, quantitation of avian beta-defensin by immunoassay, using recombinant peptide as a standard, or other such methods well known in the art, or measurement of avian beta-defensin RNA by well known hybridization techniques may serve as a diagnostic tool for infections.
Further provided is a composition comprising an amount of an isolated and purified avian beta-defensin, a variant, a derivative, or a combination thereof, in combination with a pharmaceutically acceptable carrier, such as an injectable or ingestible liquid carrier. Such a composition is useful in a method to treat or prevent a vertebrate having or at risk of having a microbial infection. Thus, the method comprises contacting a vertebrate, e.g., a bird or a human, with an amount of isolated and purified avian beta-defensin, a variant, a fragment, a derivative, or a combination thereof, effective to prevent, reduce or inhibit the infection.
Also provided is a method to prepare or identify poultry having genetically mediated superior disease resistance. The method comprises contacting a probe having at least a portion of a nucleic acid molecule of the invention with a nucleic acid sample obtained from a bird so as to form binary complexes. Then, the amount or presence of the complexes is detected or determined. Preferably such a method is used to breed poultry having certain avian beta-defensin genes. Moreover, the nucleic acid molecules of the invention may also be useful as a marker for various genetic studies. This type of marker can be used to diagnose genetic diseases which may be linked to this marker, if not directly due to a defect in the avian beta-defensin gene, as well as in restriction fragment length polymorphism (RFLP) studies for breeding purposes.
The invention also includes a method to prepare a transgenic vertebrate, such as an avian, having superior disease resistance. For example, the method comprises introducing into an avian embryo or germ cell of a bird a nucleic acid molecule of the invention so as to obtain a transgenic avian. Further provided is a transgenic vertebrate, whose cells contain a recombinant DNA sequence, wherein the recombinant DNA sequence comprises a transcriptional control sequence and a DNA segment encoding an avian beta-defensin. Preferably, the transcriptional control sequence and the DNA segment encoding an avian beta-defensin are operatively linked to each other and are integrated into the genome of the vertebrate. The DNA segment is expressed in the transgenic vertebrate so as to result in the transgenic vertebrate having increased amounts of avian beta-defensin. Methods to prepare transgenic avians are disclosed, for example, in U.S. Pat. No. 5,162,215 and WO 97/47739.
Also provided is a method for detecting nucleic acid encoding an avian beta-defensin peptide. The method comprises contacting an amount of DNA or RNA obtained from a vertebrate physiological sample which comprises cells suspected of containing DNA or RNA encoding the peptide with an amount of at least two oligonucleotides under conditions effective to amplify the DNA, or reverse transcribe the RNA and then amplify the DNA, by, e.g., a polymerase chain reaction, so as to yield an amount of amplified DNA. At least one oligonucleotide is specific for DNA encoding the avian beta-defensin. The presence of the amplified DNA is then detected or determined. Preferably, the amplified DNA is subjected to agarose gel electrophoresis prior to detection.
Thus, the invention also provides a diagnostic kit for detecting the presence of nucleic acid encoding avian beta-defensin in a sample. The kit comprises packaging containing (a) a known amount of a first oligonucleotide, wherein the first oligonucleotide consists of at least about 7 to about 50 nucleotides, preferably at least about 12 to about 15 nucleotides, and wherein the oligonucleotide has at least about 70% contiguous nucleotide sequence identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15; and (b) a known amount of a second oligonucleotide, wherein the second oligonucleotide consists of at least about 7 to about 50 nucleotides, preferably at least about 12 to about 15 nucleotides, and wherein the oligonucleotide has at least about 70% contiguous sequence identity to a nucleotide sequence which is complementary to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13 or SEQ ID NO:15.